A simplified and defined serum-free medium for cultivating fat across species

Summary Cultivated meat is a promising technology with the potential to mitigate the ethical and environmental issues associated with traditional meat. Fat plays a key role in the meat flavor; therefore, development of suitable adipogenic protocols for livestock is essential. The traditional adipogenic cocktail containing IBMX, dexamethasone, insulin and rosiglitazone is not food-compatible. Here, we demonstrate that of the four inducers only insulin and rosiglitazone are necessary in both serum-free (DMAD) and serum-containing media, with DMAD outperforming FBS. Two glucocorticoid receptor activators, progesterone and hydrocortisone, found in DMAD and FBS, affect differentiation homogeneity, without playing an essential role in activating adipogenic genes. Importantly, this protocol leads to mature adipocytes in 3D culture. This was demonstrated in both media types and in four species: ruminant and monogastric. We therefore propose a simplified one-step adipogenic protocol which, given the replacement of rosiglitazone by a food-compatible PPARγ agonist, is suitable for making cultivated fat.


Serum-free medium outperforms FBScontaining medium for differentiation
Only two inducers, rosiglitazone and insulin, are needed for serum-free adipogenesis The 1-step protocol, instead of the traditional 3-step, can be used in both 2D and 3D The simplified protocol translates across several species (cow, sheep, pig, and mouse)

INTRODUCTION
Intensive animal farming is one of the major contributors to global environmental degradation and poses great concerns on animal welfare. 1 Cultivated meat is a technology that has been proposed to overcome issues brought by conventional meat production. 2,3 The concept is based on the ability of stem cells to proliferate to significant numbers and subsequently, with appropriate stimuli, differentiate into a desired tissue. 4 The final product should resemble the taste, texture, and nutritional value of conventional meat, and for that purpose the addition of fat tissue is necessary. Fat tissue plays an essential role in body homeostasis through its energy storing and endocrine functions. [5][6][7][8] Adipocytes make up most of the fat tissue volume and are unique among cells because 95% of their entire cell body is composed of one large lipid droplet, containing high energy molecules: triglycerides. 9,10 Their unique physiological properties and the lack of a robust cytoskeleton make them important for meat taste and tenderness of meat products. [11][12][13][14] Fat tissue has been reported as the most important contributor to meat flavor across species, [15][16][17] and fatty taste or oleogustus, has been proposed as the sixth basic taste. 18 Given its role in palatability and nutritional value of a meat cut, developing an efficient protocol for fat production through adipogenic differentiation is of great importance.
The main strategies used for in vitro adipocyte differentiation are the supplementation of free fatty acids (FA) and the so-called traditional adipogenic differentiation protocol or cocktail. [19][20][21] The first one of which requires constant supply of a specific combination of free FA and can therefore be costly given the lengthy maturation period of adipocytes. An empirically derived adipogenic differentiation protocol is more commonly used and activates a signaling cascade and subsequent gene expression toward an adipocyte phenotype. 22 It consists of a combination of three or four adipogenic inducers that are added to the medium typically in three phases: induction, progression and maintenance ( Figure 1A). Induction (I) lasts from 2-4 days and signals to expanding cells to stop proliferating and start activating transcription factors which regulate adipogenesis. 23 Induction medium has insulin, IBMX, dexamethasone and, depending on the investigators, rosiglitazone. Progression medium (P) lasts 2-4 days and usually has a combination of insulin and either rosiglitazone or dexamethasone. During maintenance (M, lasts 2-24 days) either insulin alone or no inducers are added. All pathways activated by adipogenic inducers lead to peroxisome proliferator-activated receptor gamma (PPARg), through increasing its expression by the family of CCAAT ll OPEN ACCESS enhancer binding proteins (C/EBP) with insulin, dexamethasone and IBMX; or its activity by ligands such as rosiglitazone. 24 Adipogenesis is mostly studied in cell lines or primary cells from mice or human donors, and often in media containing fetal calf serum (FBS). 25,26 These monogastric species have a lipid metabolism that is different B C A Figure 1. Differentiation in serum-free versus FBS-medium (A) Simplified schematic illustration of 2D adipocyte differentiation process. Bovine SVC cells were proliferated in either serum-containing growth medium (10% FBS; GM) or serum-free growth medium (SFGM). Adipogenesis was induced in a differentiation medium with 3%FBS or a defined animal componentfree medium (DMAD). Media was changed 3 times. To assess the formation of adipocytes in response to the adipogenic cocktail in either serum or serumfree conditions, we performed fluorescent staining of the lipid droplets and nuclei, and quantified them with a high content analyzer ImageXpress Pico (HCA). Ros, rosiglitazone; and Dex, dexamethasone. (B) Representative fluorescent images of cells on day 8 of differentiation with 3%FBS or DMAD, stained with BODIPY (green lipid droplets) and Hoechst (blue nuclei). Scale bar, 100 mm. (C) Quantification of adipogenesis at day 8 and 12 with an HCA. Data are represented as mean G standard deviation (SD); error bars represent the SD of 3 independent experiments using 3 donors. Statistical significance was determined with a two-way ANOVA; NS, not significant; *p < 0.05. iScience Article from ruminants such as cows. For instance, in ruminants, lipogenesis mainly occurs in the fat tissue and the carbohydrate source for FA is acetate derived from enteric fermentation. 27 Whereas in monogastric species, glucose derived acetate is the main carbon source, and FA biosynthesis occurs primarily in the liver. 28 Differentiation of bovine adipogenic precursors using the standard adipogenic cocktail is less efficacious than in human or murine models and there is no consensus on the optimal protocol for bovine adipogenesis (Table S1). In addition, very little information on bovine adipogenesis in serum-free medium exists, which is a prerequisite for cultivated meat purposes.
Cultivated fat used for future consumption in a cultivated meat context will need to be produced with methods that are food safe. Although insulin is an endogenous protein, three of the differentiation inducers are synthetic: dexamethasone, 3-Isobutyl-1-methylxanthine (IBMX) and rosiglitazone. They were initially developed for treatment of cardiovascular/neurological, inflammatory diseases and diabetes. [29][30][31] Of these, IBMX and rosiglitazone are toxic and therefore not food-compatible. 32,33 In this study, we revisited the adipogenic protocol for bovine stromal vascular cells (SVCs) in the setting of a customized, serum-free, defined medium for adipogenic differentiation (DMAD). With a full-factorial experimental design, we investigated the necessity of adipogenic phases and compounds as well as their optimal concentrations. In addition, we questioned the relevance of distinguishing phases in adipogenesis and established a one-step differentiation protocol containing only 2 of the traditional inducers. We also explored the capacity of selected inducers to sustain adipocyte maturation in 3D long-term culture, their effect on FA profile and gene expression. To ascertain general applicability to other species we applied the changes to primary porcine and ovine adipogenic precursor cells, as well as murine (3T3-L1) cells.

Serum-free differentiation outperforms serum
To investigate differentiation with the standard adipogenic cocktail, we firstly proliferated bovine SVCs in either serum-containing growth medium (10% FBS; GM) or serum-free growth medium (SFGM). Subsequently, adipogenesis was induced by a differentiation medium that either contained serum (hereinafter referred to as 3%FBS), if proliferated in serum, or DMAD, our in-house developed serum-free differentiation medium, if proliferated in SFGM. An FBS concentration of 3% was chosen for the serum containing differentiation medium as opposed to the commonly used 10%, 21 as we found this to be the optimal concentration for bovine adipogenesis (data not shown). These results are in agreement with previous studies, which have shown that FBS may dose-dependently inhibit adipogenesis. 34 It was observed that DMAD markedly outperforms the 3%FBS condition, as seen on both day 8 and 12 ( Figure 1B), where both the percentage of positive cells and total lipid area were significantly higher in the serum-free condition ( Figure 1C). Optimization of the adipogenic cocktail for bovine SVCs in these studies was thus performed using DMAD rather than serum-containing medium. After 12 days of culture, adipogenesis was not significantly increased when compared to day 8 ( Figure 1C). Given that the additional 4 days of culture did not have a significant effect on differentiation quality, screening of adipogenic inducers during the maintenance phase/third media change was excluded. Omitting this step also allowed us to focus on developing a simplified adipogenic protocol.

Rosiglitazone and insulin sufficiently induce adipogenesis
Complete exclusion and three different concentrations of each component (rosiglitazone, insulin, IBMX and dexamethasone) were investigated, as visualized in Figure 2A. Removal of insulin or rosiglitazone from the traditional protocol (control differentiation medium; cDM) resulted in significantly decreased differentiation compared to the control, revealing their necessity ( Figures 2B and 2D). In iScience Article contrast, removing IBMX increased both the percentage of positive cells and total lipid area, whereas the removal of dexamethasone significantly increased lipid area but did not affect percentage of positive cells.
We also investigated the optimal sequence of the differentiation phases. As such, four key combinations were explored: (1) Induction followed by induction; (2) induction followed by maintenance; (3) progression followed by maintenance; and (4) progression followed by progression. It was observed that 8 days of only induction did not significantly affect differentiation; however, induction followed by maintenance reduced differentiation significantly ( Figure 2C). Whereas progression followed by maintenance increased differentiation, and progression media for the entirety of culture (hereinafter referred to as reduced Differentiation Medium; rDM) resulted in the highest level of lipid accumulation ( Figures 2C and 2D). This gives clear support that only the combination of rosiglitazone and insulin is required to induce differentiation, and that the inclusion of IBMX and dexamethasone is not needed, further implying that the distinction between induction and progression phase is no longer relevant. In addition, it re-emphasises that the maintenance medium does not increase adipogenic potential following induction or progression media.
Hydrocortisone/progesterone are necessary for homogeneous cell distribution and health during differentiation The combination of insulin and rosiglitazone is sufficient for adipogenesis as seen in Figure 2, however rosiglitazone or insulin as inducers alone were not tested. As such rDM was tested when either insulin (rDM, 0% Ins) or rosiglitazone (rDM, 0% Ros) was removed for the 8 days of culture (see illustration of experimental design in Figure 3A). The absence of rosiglitazone almost completely diminished any differentiation, whereas no insulin more than halved it ( Figures 3B and 3C). Both inducers are therefore necessary components of rDM.
As dexamethasone, a glucocorticoid receptor (GCR) activator, appeared to negatively affect adipogenesis, we further investigated the presence of hydrocortisone and progesterone (HC/PR), two endogenously present GCR activators in DMAD. We observed that removing all GCR binding molecules in 'rDM, 0% HC/PR' (removal of hydrocortisone and progesterone) resulted in significantly reduced differentiation ( Figure 3C). However, 'cDM, 0% HC/PR' (removal of dexamethasone, hydrocortisone, and progesterone but with IBMX), did not have significantly lower adipogenesis compared to cDM. In both cases however, the removal of hydrocortisone and progesterone lead to cells that differentiated in clusters and presented unhealthy morphology or even died in certain isolations ( Figures 3D and S2). This suggests that GCR binding molecules, at relatively low concentrations, are important components of adipogenic differentiation media for homogeneous differentiation, as well as for cell viability and spreading.

Reduced differentiation medium induces a high level of adipogenic markers
To verify that rDM results in adipogenic marker expression, we investigated their presence at the gene and protein level. For the gene expression panel, early differentiation-PPARg2; and adipocyte maturation markers-ADIPOQ, CIDEC and SCD were analyzed. For statistical analysis all conditions were compared to rDM. qPCR results confirmed significantly greater upregulation of all adipogenic genes except SCD in cells differentiated with DMAD rDM than with cDM ( Figure 4A). Differentiation in 3%FBS did not demonstrate significant difference in gene expression between rDM and cDM in case of SCD and ADIPOQ and was better in case of PPARg and CIDEC, suggesting that rDM can be used in combination with different media ( Figure S3). Removal of IBMX from cDM did not result in a significant difference of SCD when compared to rDM, whereas PPARg, CIDEC and ADIPOQ were downregulated. Similarly, taking out dexamethasone from cDM did not significantly decrease the expression of SCD and PPARg when compared to rDM, however CIDEC and ADIPOQ were significantly downregulated. Removing insulin and rosiglitazone on the other hand resulted in significantly lowered expression of all four genes. This confirms our initial observation that dexamethasone and IBMX are redundant in our adipogenic differentiation media.
Furthermore, to confirm adipocyte differentiation with rDM at a protein level, we investigated the presence of PPARy and CEBPa (early differentiation markers), and ACACA and PLIN1 (maturation markers). Consistent with the qPCR data, adipogenic proteins are clearly expressed ( Figure 4B). Qualitatively, cDM appears to result in lower amounts of adipogenic proteins than rDM overall ( Figures 4B and S4). Together, this data indicates that cells can differentiate into mature adipocytes with rDM, warranting it as an adipogenic cocktail for cultivated fat. iScience Article (3%FBS) or serum-free (DMAD) conditions, as illustrated in Figure 5A. Differentiation quality was assessed via confocal microscopy, glycerol release and lipidomic profiling.
At week 1, 2D adipocyte cultures appear to have more lipid droplets than 3D alginate microfibers ( Figure S5A), likely because of a lack of interaction between cells in the non-functionalized alginate. However, given that 2D adipocyte cultures have the tendency to detach in long-term cultures ($28 days), 3D constructs are required.
Assessment at week 1 of the microfiber culture visibly shows that the cells start to differentiate with higher efficiency with rDM when compared to cDM ( Figure S5A). This trend continues onto week 4, where rDM results in an increased number of differentiating cells and bigger lipid droplet diameter than cDM in both DMAD and 3%FBS ( Figure 5B). Cells within the microfibers differentiated with both rDM and cDM showed a strong signal for PLIN1 ( Figure 5C). Glycerol release, typically used to study triglyceride/FA cycling, revealed glycerol concentration increased with time of culture in both rDM and cDM, with no significant difference between the two conditions ( Figure 5D).
With lipidomic profiling we confirmed the qualitative observations in Figure 5B: rDM significantly outperforms cDM in both serum and serum-free media in total lipid content ( Figures 5E and S5C). Subsequently we compared the triglyceride composition of conventional and cultivated fat ( Figure S6). Bovine fat had a significantly higher amount of palmitic (16:0) and linoleic acid (18:2) compared to all cultivated samples ( Figure 5E). Depending on the in vitro condition, cultivated fat contained either a similar amount of stearic acid (18:0) (cDM DMAD), lower (rDM DMAD/FBS), or higher amounts (rDM FBS) compared to bovine fat. Oleic acid (18:1) is significantly higher in cultivated fat compared to bovine fat. Long chain polyunsaturated fatty acids (PUFAs) are absent in bovine fat but detectable in fat cultured with DMAD, in particular in the cDM condition.

Transcriptomic investigation of the rDM inducers
To characterize and compare the changes at the gene expression level that each essential inducer has on adipogenesis, we have differentiated bovine SVC with rDM or rDM deprived from insulin, rosiglitazone, and HC/PR, and performed RNAseq on these samples. We observed that taking out rosiglitazone and insulin resulted in large changes in gene expression levels when compared to rDM, whereas taking out HC/ PR showed a more comparable pattern to rDM and less changes at the gene expression level ( Figure 6A). This is in line with the phenotypic effect on differentiation that insulin and rosiglitazone exhibit on bovine SVC. Enriched gene ontology (GO) terms indicated the crucial role of insulin and rosiglitazone in upregulating the expression of genes related to adipocyte phenotype and highlighted their overlapping effect on gene expression ( Figure 6B). rDM compared to rDM without insulin results in 57 significantly enriched upregulated GO terms related mainly to FA and lipid metabolism, oxidative phosphorylation, and cholesterol metabolism ( Figure S7). Rosiglitazone results in 38 GO terms mainly involved in lipid metabolism and HC/PR in 10 enriched GO terms related to aerobic respiration and FA metabolic processes. From the list of shared GO terms, we focused on the most prominently present terms indicated in Figure 7B. With multiple markers we observed that rDM shows upregulation of FA storing or metabolism related genes (CIDEA, SCD, ELOVL6, FASN) as well as FA and cholesterol transportation (APOE) and numerous genes involved in aerobic respiration (COX6A1). Genes which are reported to be involved in the transmission of the signal generated by adipogenic inducers (FOXO1, CREB1, NR3C1) did not show significant upregulation ( Figure S7). In addition, we observed that cells differentiated with rDM do not show expression of the brown fat marker UCP-1, indicating that our media selectively induces white adipogenesis over brown.

rDM robustly supports differentiation across various species
Studies on monogastric species (mouse and human) have been crucial for understanding adipogenesis and developing traditionally used protocols (cDM in medium containing serum). However, these protocols do not allow for efficient in vitro differentiation of ruminant species such as cows, possibly due to significant differences between monogastric and ruminant physiology and lipid metabolism. Contrarily rDM performs well in iScience Article differentiating bovine SVCs, thus for understanding whether reduced inducers are species specific and if they could be used for application in other cultivated meat products, we have assessed the translation of our onestep protocol to another ruminant animal (sheep) and two monogastric species (pig and mouse). For this we compared the capacity of rDM to efficiently differentiate ovine and porcine FAP cells, as well as murine 3T3-L1 cells. Cells derived from the monogastric species, pig and mouse, differentiated efficiently in cDM 3%FBS (Figure 7), whereas sheep cells did not, much like cow cells ( Figure 5). Although cells from all three species, conversely, underwent high levels of adipogenesis with rDM DMAD. According to these results, rDM is not species specific, as opposed to the traditional protocol using FBS and the four conventional inducers.

DISCUSSION
For initiating adipogenesis in cell culture, a cocktail of inducers containing insulin, IBMX and dexamethasone, and frequently rosiglitazone has been reported as necessary. Predominantly these studies used mouse or human cell lines in serum-containing media. Here, we report superior adipogenesis in bovine SVCs in serum-free, chemically defined, differentiation medium in 2D and 3D. In this medium, which contains a relatively low amount of steroids, dexamethasone has no additional adipogenic activity. IBMX, a substance that is toxic for humans and therefore not food-compatible, could also be eliminated from the adipogenic cocktail, leaving only insulin and rosiglitazone as the required adipogenic inducers.
Understanding of adipogenic differentiation started with work on mouse embryonic fibroblasts and development of the self-differentiating 3T3-L1 cell line. 35,36 With addition of insulin, IBMX and dexamethasone, differentiation time shortened from 4 weeks to 6-7 days, and adipogenesis increased presenting up to 90% positive cells. 37 Phenotype reproducibility and lack of donor-to-donor variability of 3T3-L1 was of great importance in shaping and accelerating the knowledge behind adipocyte differentiation. However, the protocol has been adopted across different species and cell types with varying efficacy. For example, mouse embryonic cell lines including 3T3-442A, 1246, Ob1771 require different inducers for optimal differentiation 26,38,39 and the differentiation of ruminant adipogenic precursors using the traditional protocol shows low efficacy.
Most adipogenic differentiation studies were performed using FBS. Besides batch-to-batch variability, ethical concerns and unknown composition, serum contains molecules involved in adipogenic differentiation (insulin z10 mU/mL, cortisone z up to 54 ng/mL, progesterone z up to 80 ng/mL), leading to difficulties in studying the inducers. [40][41][42] Although several investigators have explored inducer necessity in serum, 21,43,44 the few reports on efficient differentiation in a serum-free environment [45][46][47][48][49] were limited to the standard adipogenic protocol. Using full factorial design in serum-free medium (Table S3) we showed that IBMX and dexamethasone can be excluded from the differentiation media.
IBMX is a phosphodiesterase inhibitor which increases intracellular cAMP, known to play a key role in 3T3-L1 differentiation. 50 In our hands, supplementation of IBMX during induction did not benefit differentiation; on the contrary, increasing its concentration or exposure time resulted in cell detachment and death, in line with previous studies. 50,51 IBMX was long reported to act on adipogenesis through the increase of C/EBPb expression via cAMP response element-binding protein (CREB). 52,53 Later, it was discovered that cAMP-induced signaling overlaps with insulin-induced signaling 54,55 supporting our observation that IBMX does not have an additive effect as an inducer. iScience Article Elevated local levels of glucocorticoids in fat tissue are reported to result in increased obesity and metabolic syndrome, 56 however these observations are conflicting between species or tissue depots. 57,58 Dexamethasone is a synthetic glucocorticoid which binds to the GCR and acts on CEBPd and CEBPb. [59][60][61] Its endogenous alternatives hydrocortisone and progesterone, 62 which are present in FBS, are reported to have lower activity on GCR 37 and differentiation potential compared to dexamethasone; dexamethasone is therefore still supplemented across adipogenic protocols. Of interest, we observed that leaving out dexamethasone did not have an impact on differentiation quality. However, because DMAD contains hydrocortisone and progesterone (at concentrations comparable to 10% FBS and slightly higher than 3% FBS -up to 4.47 nM HC and 7.6 nM PR in 3% FBS, versus 25 nM HC and 17 nM PR in DMAD 40,42 ), omitting only dexamethasone hindered the assessment of GC signaling during adipogenesis. After depleting DMAD from all the GCR activators, we observed lower differentiation, with cells presenting unhealthy morphology, lower cell numbers and differentiation in islands. However, excluding or including hydrocortisone and progesterone from rDM did not result in a large change at the adipogenic gene expression level. The importance of hydrocortisone in cell attachment, spreading and proliferation of SVC has been documented in several serum-free environments, 63,64 our findings similarly imply that GCR-induced signaling might be more important for cell survival related mechanisms than adipogenesis. 61,65,66 The inclusion of dexamethasone and IBMX therefore does not appear necessary for adipocyte differentiation.
Insulin was the first adipogenic stimulator discovered and in the absence of other inducers it is reported to ameliorate differentiation of 3T3-L1 cells in a concentration dependent manner. 67 In our culture, removing insulin from cDM or rDM resulted in few differentiating cells containing small lipid droplets. The presence of insulin showed significant upregulation of a multitude of adipogenic and lipogenic genes, it also indicated its importance in cholesterol metabolism and oxidative phosphorylation during differentiation. A great overlap of enriched upregulated GO terms when insulin or a PPARg agonist are supplemented points out the converging mechanisms that these two have in promoting adipocyte differentiation. Our observations are in line with numerous previous studies in different species where insulin importance was demonstrated in vitro and in vivo. 68,69 The beneficial effects of PPARg activation on adipogenesis were observed when the PPARg agonist, indomethacin, induced high levels of differentiation in 3T3-L1 cells. 37,70 Subsequently, highly specific PPARg agonists were developed for the treatment of diabetes, 71 and notably rosiglitazone emerged as a potent adipogenic stimulator. 72 Similarly, we observed that rosiglitazone effectively induced differentiation, whereas its absence resulted in minimal adipogenesis. Unlike other factors, in 2D culture it is sufficient to induce adipogenesis in a relatively small number of cells in the absence of all other inducers. However, in our 3D system, DMAD negative control showed a discrete number of cells containing large lipid droplets ( Figure S5B). This is contrary to our observations in the 2D short-term culture, where the negative control (containing insulin and HC/PR, but no PPARg agonist) did not differentiate. Differentiation is possibly driven by our media composition and the presence of lipid concentrate in DMAD, where fatty acids such as linoleic and oleic are known to have a weak PPARg activating function. 19,20 Stronger PPARg agonists, such as rosiglitazone, are likely necessary to increase the percentage of differentiating cells. The pro-adipogenic effect of rosiglitazone was supported at the gene expression level where the highest number of differentially expressed genes was present when rDM was deprived from rosiglitazone. Therefore, a PPARg agonist is necessary to induce adipogenesis of bovine SVCs.
For an appealing cultivated meat product, besides a low-cost, animal component-free, safe media, cultivated fat should mimic the nutritional and sensory qualities of conventional beef fat. For this purpose, we performed lipidomic investigation of cultivated fat and cow derived fat. We focused on the FA composition within triglycerides iScience Article because FA species have a major effect on the sensory and nutritional value of fat. 15 Stearic acid has been reported to greatly impact the firmness and the melting point of bovine fat. 14,73 Cultivated fat differentiated with rDM DMAD did not show significant differences in stearic acid compared to conventional bovine fat. Oleic acid has been reported to greatly impact taste and high amounts improve sensory quality. 74 Of interest, in vitro grown adipocytes from all four media have much higher amounts of oleic acid compared to bovine fat, an observation with unclear underlying mechanism. PUFAs have a major impact on human health. It has been well documented across the literature that bovine fat does not contain long chain PUFA within neutral lipids. 14 In cells grown with DMAD several species of PUFA were present. rDM did not show significantly higher amounts of EPA and DHA however cDM had higher amounts of all the members of long chain PUFA when compared to bovine fat. This exciting result suggests that in vitro grown adipocytes, if cultured with food compatible signaling molecules, can result in healthier fat for human consumption.
In addition, to the best of our knowledge, our reduced defined medium is the first to demonstrate high differentiation level across four species and is comparable (monogastric) or better (ruminant) than the traditional cocktail in FBS containing medium. Having a medium able to differentiate adipogenic precursors originating from different tissues 75 and domestic animals is of great importance for making cultivated fat.
We have yet to determine how DMAD is able to overcome physiological differences reported during adipogenesis between species, in vivo and in FBS.
With our study we established a new, simplified one-step adipogenic protocol with a reduced number of adipogenic inducers and phases. The most important inducers are rosiglitazone and insulin, whereas GCR activators ameliorate differentiation quality. Our newly developed protocol in an animal component-free defined medium with the reduced number of inducers can be used across two monogastric and two ruminant species for high quality differentiation. This work has narrowed the search for finding food safe inducers to only a PPARg agonist before establishing a compatible media for cultivated fat production.

Limitations of the study
This study investigated changes at the gene expression level that each inducer has on adipogenesis. To add to the understanding of signaling pathways activated by each inducer within the new rDM medium, future work iScience Article should focus also on the changes occurring at a protein level. In addition, we demonstrate that although the rDM induces and sustains long-term 3D differentiation, the timings and necessity of each rDM inducer during the entire 28-day culture were not investigated. Lastly, rosiglitazone is not a food compatible component and thus cannot be used for cultured fat production, requiring an alternative. These questions would form the basis of future studies into rDM.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   Stromal vascular cells (SVC) cells were isolated from the subcutaneous brisket fat of Belgian Blue cattle (both male and female, aged 1 to 7 years) as previously described. 19,75 Briefly, fat tissue was minced and dissociated with collagenase (CLSAFA, Worthington; 2 h, 37 C). Cell pellets containing SVC and the supernatant containing mature adipocytes were washed with PBS, centrifuged again and pellets were filtered through a 100 mm cell strainer. Filtered cells were resuspended in growth medium (GM; see Table S3) or in defined, in-house developed, serum-free proliferation medium (SFGM; see Table S3) and seeded (10 grams of initial starting tissue per 75 cm 2 ) in cell culture flasks for propagation.

Isolation of ovine and porcine adipogenic precursor cells
Fresh ovine and porcine skeletal muscle was obtained from a registered abattoir according to national guidelines on animal tissue handling. Muscle-derived adipogenic precursors (referred to as Fibro-adipogenic progenitor cells (FAP) from here on) were isolated as previously described. 76 Semitendinosus muscle was minced and dissociated with collagenase (CLSAFA, Worthington; 1 h, 37 C). Cell slurries were filtered through a 100 mm cell strainer and incubated in ammonium-chloride-potassium (ACK) erythrocyte lysis buffer (1 min, room temperature (RT)). Cells were resuspended in GM, filtered through a 40 mm strainer prior to culture and seeded (10 grams of initial starting tissue per 75 cm 2 ) in cell culture flasks. Murine, porcine, and ovine cells were grown in GM.

Cell culture
Cells were grown on tissue culture-coated culture dishes (Corning) in GM or in SFGM. 77 For serial expansion, cells were cultured until 80-90% confluent, incubated with preheated trypsin-EDTA 1X (Gibco, 25300-062) at 37 C for 4 min. Trypsin was inactivated with GM/SFGM. Detached cells were then centrifuged at 350 3 g for 5 min, resuspended in 1 mL and mounted on a counting chamber using trypan blue. Cell counting and viability estimation were performed with an automatic cell counter Countess (Thermo Fisher). For serial passaging, cells were seeded at a density of 5310 3 cells/cm 2 , incubated at 37 C, 5% CO 2 and passaged at 80% of confluency.

Cell differentiation into adipocyte lineage
SVCs were differentiated at passage 2, 3 and 4, porcine (Sus scrofa domesticus) and ovine (Ovis aries) FAPs at passage 2, and 3T3-L1 cells at passage 7. For 2D differentiation cells were seeded at a density of 3310 4 cells/cm 2 and proliferated for 1 day. Following this, either DMAD (Table S3) or 3%FBS differentiation medium (Table S3) were added to the cells, supplemented with different combinations/concentrations/ timings of adipogenic inducers. In all cases of differentiation, cells proliferated in serum (GM) were differentiated in 3%FBS differentiation medium and cells proliferated in serum-free medium (SFGM) were differentiated in DMAD medium. Adipogenic inducers consist of insulin 10 mg/mL (Sigma; I0516); dexamethasone 1 mM (Sigma; D4902); IBMX 0.5 mM (Sigma; I5879) and rosiglitazone 5 mM (Sigma; R2408). First medium change containing all four inducers is referred to as induction medium, second medium change containing rosiglitazone and insulin as progression medium and third medium change without inducers as maintenance medium. Medium was changed every 4 days. The media containing all four components in the induction, insulin and rosiglitazone in progression, and maintenance with only insulin was our control (cDM; based on a protocol by Zhou et al., 2010 78 ). To investigate inducer necessity, we did a full factorial design, that is we have tested all the possible inducer combinations (Figures 2 and S1) in the first two media changes (no software was used for the design). All 2D differentiation experiments were carried out in a Falcon 96-well plate with a well surface of 0.34 cm 2 . Details of the 3D differentiation cultures are given below.

Adipogenic microfiber cultures
SVCs were resuspended at a concentration of 6310 7 cells/mL in the respective differentiation medium. The cell suspension was mixed at a 1:1 v/v ratio with 1% alginate T1 H2O, to reach a final concentration of 0.5% ll OPEN ACCESS iScience Article RNA sequencing and analysis SVCs were seeded in 2D at 3310 4 cells/cm 2 , proliferated for a day in SFGM and differentiated for 12 days in rDM. mRNA was extracted using the Dynabeadsâ mRNA DIRECTä MicroKit (Thermo Fisher). Subsequently 1 ng of mRNA was reversed transcribed into cDNA using the Maxima H Minus Reverse Transcriptase (Thermo Fisher) and the primers from the PCR-cDNA Barcoding kit (SQK-PCB109, Oxford Nanopore Technologies). The cDNA was then barcoded using the same PCR-cDNA barcoding kit, which was then also used to finish the library prep. The libraries were sequenced using two R9.4.1 flowcells (Oxford Nanopore Technologies) on a GridION (Oxford Nanopore Technologies) for 48 hours.
Basecalling was performed using Guppy version 5.0.16 in the Super-accurate setting (minimal q-score threshold of 10) with automatic de-multiplexing and barcode removal.
The filtered reads were mapped to the bovine transcriptome (ARS-UCD1.2) using Minimap2 79 with the default settings for Oxford Nanopore reads (-ax map-ont), and then subsequently quantified using Salmon. 80 The data analysis was performed using DEseq2, 81 with tximport 82 for importing the quantified data from Salmon. Batch correction for the 3 separate batches of mRNA extraction and cDNA making was done using Combat-Seq from the SVA package. 83,84 After differential gene expression analysis, gene set enrichment analysis for GO terms 85,86 and WikiPathways 87 was performed using the Piano R package 88 using the following 7 methods: mean, median, sum, GSEA, PAGE, stouffer's and tail Strength with 10,000 permutations each and a gene set size limit of min 10 and max 500. The median adjusted p Value across all methods is reported for each gene set as the final p Value. All steps were performed in-house, including the use of the Oxford nanopore and the data analysis.

Lipidomic analysis
Cultured fat samples (10-30 mg) were collected for lipidomic analysis. The samples were then sent to Lipometrix, the KU Leuven lipidomics core facility, to carry out the processing and analysis of lipids as described here. Lipids were extracted using a modified Bligh-Dyer protocol and analysed by hydrophilic interaction liquid chromatography mass spectrometry (HILIC LC-MS/MS). 89 Lipid quantities were normalised to the amount of DNA present within the respective sample. For data analysis, peak integration was performed with MultiQuantTM (version 3.0.3). Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1) and quantified based on internal standard signals as per the Lipidomics Standards Initiative (LSI).

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analysis was performed using Prism 9.1.0 (GraphPad). For comparing two groups unpaired t-test was performed. Analysis of three or more groups was performed using one-way ANOVA with Bonferroni's multiple comparisons test against indicated control(s). Analysis of three or more groups with two independent variables was performed using a two-way ANOVA with Bonferroni's multiple comparison test against indicated control(s). Additional details on the statistical analysis of each experimental group can be found in the figure legends. Error bars in all figures represent the mean G standard deviation (SD). N represents the number of donor animals. Technical replicates (multiple wells) were averaged, and statistical analysis was performed on biological replicates and replicates of individual experiments.